Method for promoting neovascularization

ABSTRACT

The present invention relates to a method for enhancing angiogenic activity to promote neovascularization comprising administering to a subject a formulation comprising a synergistically effective amount of a TWEAK agonist and an angiogenic factor.

FIELD OF INVENTION

The present invention relates to a method for enhancing angiogenicactivity to promote neovascularization.

BACKGROUND OF THE INVENTION

Growth of microvasculature, or angiogenesis, involves endothelial cell(EC) proliferation, migration, differentiation, and structuralorganization into new vessels.

Angiogenic regulators induce changes in endothelial cells (EC) at avariety of levels, including their proliferative, migratory, secretory,and adhesive properties, and may do so through their action on ECs orother cell types (Kumar et al, 1998, Int. J. Oncology 12:749-757;Bussolino et al., 1997, Trends in Biochem, 22:251-256). Several TNFfamily ligands previously have been implicated in the process ofangiogenesis, namely TNFα, FasL and TWEAK.

SUMMARY OF THE INVENTION

TWEAK, a novel member of the TNF ligand family, may promote angiogenesisbased on its ability to induce IL-8 production by several epithelialtumor cell lines, proliferation in various human EC and aortic smoothmuscle cells under reduced growth factor conditions, and stimulation ofan angiogenic response when implanted in rat corneas. Herein, we furthercharacterize the angiogenic potential of TWEAK, demonstrating that TWEAKsynergizes with Fibroblast Growth Factor (FGF) to induce proliferationand migration of EC's. While TWEAK weakly promotes EC survival, thesynergistic effect of TWEAK and FGF on EC proliferation appears to bedue to potentiation of cell division rather than decreased cell death.TWEAK also did not detectably alter the expression of receptors for FGFor VEGF, or expression of the integrins α₁, α₅, α_(v), β₁, or β₃. Theability of TWEAK to induce ECs to form capillaries in the absence ofother cell types was demonstrated in a 3D fibrin gel matrix where,strikingly, TWEAK induced the morphogenesis of lumens in invading,bFGF-dependent EC cords. Our findings further distinguish TWEAK fromother TNF family ligands, demonstrating its ability to promoteangiogenesis at multiple discrete stages.

One aspect of the present invention is a method for enhancingendothelial cell proliferation in an in vitro culture comprising addingto said culture, a formulation consisting essentially of asynergistically effective amount of a TWEAK agonist and an angiogenicfactor.

A second aspect of the present invention is a method for enhancingangiogenic activity in a mammal to promote neovascularization comprisingthe step of administering to said mammal a formulation comprising asynergistically effective amount of a TWEAK agonist and an angiogenicfactor sufficient to promote neovascularization. A preferred embodimentis the use of bFGF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effect of TWEAK on bFGF-dependent HUVECproliferation.

FIG. 2 illustrates the effect of TWEAK on HUVEC death.

FIG. 3 illustrates the effect of TWEAK on bFGF-dependent HUVECmigration.

FIG. 4 depicts the synergistic effect of TWEAK and bFGF on capillarytube formation.

DETAILED DESCRIPTION

As used herein the terms, “angiogenesis,” “revascularization,”“increased collateral circulation,” and “regeneration of blood vessels”are considered as synonymous.

“Angiogenesis” is defined as any alteration of an existing vascular bedor the formation of new vasculature which benefits tissue perfusion.This includes the formation of new vessels by sprouting of endothelialcells from existing blood vessels or the remodeling of existing vesselsto alter size, maturity, direction or flow properties to improve bloodperfusion of tissues.

A therapeutic is said to have “therapeutic efficacy” in modulatingangiogenesis and an amount of the therapeutic is said to be a“angiogenic modulatory amount”, if administration of that amount of thetherapeutic is sufficient to cause a significant modulation (i.e.,increase or decrease) in angiogenic activity when administered to asubject (e.g., an animal model or human patient) needing modulation ofangiogenesis.

The term angiogenic factor refers to factors which promote theangiogenic process, including but not limited to the following phases ofthe process, ie. the degradation of the extracellular matrix, cellproliferation, cell migration and structural organization (Kumar et al,1998, Int. J. Oncology 12:749-757; Bussolino et al., 1997, Trends inBiochem, 22:251-256). Angiogenic factors include but are not limited tofibroblast growth factor (bFGF), acidic FGF (aFGF), FGF-5, vascularendothelial growth factor isoforms (VEGF), angiopoietin-1 (Ang-1) andangiopoietin-2 (Ang-2), Platelet-derived endothelial cell growth factor(PD-ECGF), hepatocyte growth factor (HGF), interleukin-8 (IL-8),granulocyte-colony stimulating factor (G-CSF), placental growth factor,proliferin, B61, soluble vascular cell adhesion molecular-1, solubleE-selection, 12-hydrozyeicosatetraenoic acid, Tat protein of HIV-1,angiogenin, TNFα, FasL, Transforming growth factor-β.

As used herein, the ability of TWEAK to act synergistically with anotherangiogenic factor means that the combination of TWEAK and the angiogenicfactor induce a response that is greater than the sum of the responsesto either agent alone, as measured in one or more in vitro assays whichmeasure stages of the angiogenic process. These include but are notlimited to endothelial cell survival, proliferation, migration, orcapillary tube formation, as described herein.

The term “pharmaceutically acceptable” when referring to a natural orsynthetic substance means that the substance has an acceptable toxiceffect in view of its much greater beneficial effect, while the relatedthe term, “physiologically acceptable,” means the substance hasrelatively low toxicity.

As used herein, the term “antibody homolog” includes intact antibodiesconsisting of immunoglobulin light and heavy chains linked via disulfidebonds. The term “antibody homolog” is also intended to encompass a TWEAKtherapeutic comprising one or more polypeptides selected fromimmunoglobulin light chains, immunoglobulin heavy chains andantigen-binding fragments thereof which are capable of binding to one ormore antigens (i.e., TWEAK or patched). The component polypeptides of anantibody homolog composed of more than one polypeptide may optionally bedisulfide-bound or otherwise covalently crosslinked. Accordingly,therefore, “antibody homologs” include intact immunoglobulins of typesIgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the lightchains of the immunoglobulin may be of types kappa or lambda or portionsof intact antibodies that retain antigen-binding specificity, forexample, Fab fragments, Fab′ fragments, F(ab′)2 fragments, F(v)fragments, heavy chain monomers or dimers, light chain monomers ordimers, dimers consisting of one heavy and one light chain, and thelike.

As used herein, a “humanized antibody homolog” is an antibody homolog,produced by recombinant DNA technology, in which some or all of theamino acids of a human immunoglobulin light or heavy chain that are notrequired for antigen binding have been substituted for the correspondingamino acids from a nonhuman mammalian immunoglobulin light or heavychain. A “human antibody homolog” is an antibody homolog in which allthe amino acids of an immunoglobulin light or heavy chain (regardless ofwhether or not they are required for antigen binding) are derived from ahuman source.

An “amino acid” is a monomeric unit of a peptide, polypeptide, orprotein. There are twenty amino acids found in naturally occurringpeptides, polypeptides and proteins, all of which are L-isomers. Theterm also includes analogs of the amino acids and D-isomers of theprotein amino acids and their analogs.

The term “bioavailability” refers to the ability of a compound to beabsorbed by the body after administration. For instance, a firstcompound has greater bioavailability than a second compound if, whenboth are administered in equal amounts, the first compound is absorbedinto the blood to a greater extent than the second compound.

An “expression vector” is a polynucleotide, such as a DNA plasmid orphage (among other common examples) which allows expression of at leastone gene when the expression vector is introduced into a host cell. Thevector may, or may not, be able to replicate in a cell.

The phrase “extracellular signaling protein” means any protein that iseither secreted from a cell, or is associated with the cell membrane,and upon binding to the receptor for that protein on a target cell,triggers a response in the target cell.

A “functional equivalent” of an amino acid residue is (i) an amino acidhaving similar reactive properties as the amino acid residue that wasreplaced by the functional equivalent; (ii) an amino acid of a ligand ofa polypeptide of the invention, the amino acid having similar propertiesas the amino acid residue that was replaced by the functionalequivalent; (iii) a non-amino acid molecule having similar properties asthe amino acid residue that was replaced by the functional equivalent.

“Heterologous promoter” as used herein is a promoter which is notnaturally associated with a gene or a purified nucleic acid.

“Homology” and “identity” each refer to sequence similarity between twopolypeptide sequences, and both ‘homology and ‘identity’ are usedinterchangeably in this disclosure. Homology can be determined bycomparing a position in each sequence which may be aligned for purposesof comparison. When a position in the compared sequence is occupied bythe same amino acid residue, then the polypeptides can be referred to asidentical at that position; when the equivalent site is occupied by thesame amino acid (e.g., identical) or a similar amino acid (e.g., similarin steric and/or electronic nature), then the molecules can be referedto as homologous at that position. A percentage of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences. An “unrelated” or “non-homologous”sequence shares less than 40 percent identity, though preferably lessthan 25 percent identity, with a sequence of the present invention.

For instance, if 6 of 10 of the positions in two sequences are matchedor are homologous, then the two sequences are 60% homologous. By way ofexample, the DNA sequences CTGACT and CAGGTT share 50% homology (3 ofthe 6 total positions are matched). Generally, a comparison is made whentwo sequences are aligned to give maximum homology. Such alignment canbe provided using, for instance, the method of Needleman et al., J. MolBiol. 48: 443-453 (1970), implemented conveniently by computer programsdescribed in more detail below. Homologous sequences share identical orsimilar amino acid residues, where similar residues are conservativesubstitutions for, or “allowed point mutations” of, corresponding aminoacid residues in an aligned reference sequence. In this regard, a“conservative substitution” of a residue in a reference sequence arethose substitutions that are physically or functionally similar to thecorresponding reference residues, e.g., that have a similar size, shape,electric charge, chemical properties, including the ability to formcovalent or hydrogen bonds, or the like. Particularly preferredconservative substitutions are those fulfilling the criteria defined foran “accepted point mutation” in Dayhoff et al., 5: Atlas of ProteinSequence and Structure, 5: Suppl. 3, chapter 22: 354-352, Nat. Biomed.Res. Foundation, Washington, D.C. (1978).

“Percent homology/identity” of two amino acids sequences or two nucleicacid sequences is determined using the alignment algorithm of Karlin andAltschul (Proc. Nat. Acad. Sci., USA 87: 2264 (1990) as modified inKarlin and Altschul (Proc. Nat. Acad. Sci., USA 90: 5873 (1993). Such analgorithm is incorporated into the NBLAST or XBLAST programs of Altschulet al., J. Mol. Biol. 215: 403 (1990). BLAST searches are performed withthe NBLAST program, score=100, wordlength=12, to obtain nucoetidesequences homologous to a nucleic acid of the invention. BLAST proteinsearches are performed with the XBLAST program, score=50, wordlength=3,to obtain amino acid sequences homologous to a reference polypeptide. Toobtain gapped alignments for comparisons, gapped BLAST is used asdescribed in Altschul et al., Nucleic Acids Res., 25: 3389 (1997). Whenusing BLAST and Gapped BLAST, the default parameters of the respectiveprograms (XBLAST and NBLAST) are used. See http://www/ncbi.nlm.nih.gov

The term “hydrophobic” refers to the tendency of chemical moieties withnonpolar atoms to interact with each other rather than water or otherpolar atoms. Materials that are “hydrophobic” are, for the most part,insoluble in water. Natural products with hydrophobic properties includelipids, fatty acids, phospholipids, sphingolipids, acylglycerols, waxes,sterols, steroids, terpenes, prostaglandins, thromboxanes, leukotrienes,isoprenoids, retenoids, biotin, and hydrophobic amino acids such astryptophan, phenylalanine, isoleucine, leucine, valine, methionine,alanine, proline, and tyrosine. A chemical moiety is also hydrophobic orhas hydrophobic properties if its physical properties are determined bythe presence of nonpolar atoms.

The phrase “internal amino acid” means any amino acid in a peptidesequence that is neither the N-terminal amino acid nor the C-terminalamino acid.

“Isolated” (used interchangeably with “substantially pure”) when appliedto nucleic acid i.e., polynucleotide sequences that encode polypeptides,means an RNA or DNA polynucleotide, portion of genomic polynucleotide,cDNA or synthetic polynucleotide which, by virtue of its origin ormanipulation: (i) is not associated with all of a polynucleotide withwhich it is associated in nature (e.g., is present in a host cell as anexpression vector, or a portion thereof); or (ii) is linked to a nucleicacid or other chemical moiety other than that to which it is linked innature; or (iii) does not occur in nature. By “isolated” it is furthermeant a polynucleotide sequence that is: (i) amplified in vitro by, forexample, polymerase chain reaction (PCR); (ii) synthesized chemically;(iii) produced recombinantly by cloning; or (iv) purified, as bycleavage and gel separation.

“Isolated” (used interchangeably with “substantially pure”) when appliedto polypeptides means a polypeptide or a portion thereof which, byvirtue of its origin or manipulation: (i) is present in a host cell asthe expression product of a portion of an expression vector; or (ii) islinked to a protein or other chemical moiety other than that to which itis linked in nature; or (iii) does not occur in nature, for example, aprotein that is chemically manipulated by appending, or adding at leastone hydrophobic moiety to the protein so that the protein is in a formnot found in nature. By “isolated” it is further meant a protein thatis: (i) synthesized chemically; or (ii) expressed in a host cell andpurified away from associated and contaminating proteins. The termgenerally means a polypeptide that has been separated from otherproteins and nucleic acids with which it naturally occurs. Preferably,the polypeptide is also separated from substances such as antibodies orgel matrices (polyacrylamide) which are used to purify it.

A “protein” is any polymer consisting essentially of any of the 20 aminoacids. Although “polypeptide” is often used in reference to relativelylarge polypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and is varied.The term “protein” as used herein refers to peptides, proteins andpolypeptides, unless otherwise noted.

The terms “peptide(s)”, “protein(s)” and “polypeptide(s)” are usedinterchangeably herein. The terms “polynucleotide sequence” and“nucleotide sequence” are also used interchangeably herein.

“Recombinant,” as used herein, means that a protein is derived fromrecombinant, mammalian expression systems.

Thus, “substantially pure nucleic acid” is a nucleic acid which is notimmediately contiguous with one or both of the coding sequences withwhich it is normally contiguous in the naturally occurring genome of theorganism from which the nucleic acid is derived. Substantially pure DNAalso includes a recombinant DNA which is part of a hybrid gene encodingadditional TWEAK sequences.

The amounts of a TWEAK agonist and angiogenic factor required to beeffective in enhancing angiogenic activity for promotingneovascularization will, of course, vary with the individual beingtreated and is ultimately at the discretion of the physician. Thefactors to be considered include the condition of the patient beingtreated, the efficacy of the particular TWEAK agonist being used, thenature of the formulation, and the patient's body weight. While itpossible to administer and a TWEAK agonist simultaneously, it is alsocontemplated that angiogenic factor can be given as a bolus beforestarting the infusion of the TWEAK agonist. It is also contemplated thatangiogenic factor can be administered after the infusion of the TWEAKagonist.

TWEAK agonists include those taught in WO98/05783, WO98/35061 andWO99/19490 all of which are incorporated herein by reference. Such TWEAKagonists include soluble recombinant TWEAK protein.

“Standard hybridization conditions” refer to salt and temperatureconditions substantially equivalent to 0.5×SSC to about 5×SSC and 65° C.for both hybridization and wash. The term “standard hybridizationconditions” as used herein is therefore an operational definition andencompasses a range of hybridization conditions. Nevertheless, for thepurposes of this present disclosure “high stringency” conditions includehybridizing with plaque screen buffer (0.2% polyvinylpyrrolidone, 0.2%Ficoll 400; 0.2% bovine serum albumin, 50 mM Tris-HCl (pH 7.5); 1 MNaCl; 0.1% sodium pyrophosphate; 1% SDS); 10% dextran sulfate, and 100μg/ml denatured, sonicated salmon sperm DNA at 65° C. for 12-20 hours,and washing with 75 mM NaCl/7.5 mM sodium citrate (0.5×SSC)/1% SDS at65° C. “Low stringency” conditions include hybridizing with plaquescreen buffer, 10% dextran sulfate and 110 μg/ml denatured, sonicatedsalmon sperm DNA at 55° C. for 12-20 hours, and washing with 300 mMNaCl/30 mM sodium citrate (2.0×SSC)/1% SDS at 55° C. See also CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. New York,Sections 6.3.1-6.3.6, (1989).

A “therapeutic composition” as used herein is defined as comprising thetherapeutics of the invention and other biologically compatibleingredients. The therapeutic composition may contain excipients such aswater, minerals and carriers such as protein.

“Wild type” means the naturally-occurring polynucleotide sequence of anexon of a protein, or a portion thereof, or protein sequence, or portionthereof, respectively, as it normally exists in vivo.

Practice of the present invention will employ, unless indicatedotherwise, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, protein chemistry, andimmunology, which are within the skill of the art. Such techniques aredescribed in the literature. Unless stipulated otherwise, all referencescited in the Detailed Description are incorporated herein by reference.

A. Production of Fragments and Analogs

Fragments of an isolated protein (e.g., fragments of TWEAK) can also beproduced efficiently by recombinant methods, by proteolytic digestion,or by chemical synthesis using methods known to those of skill in theart. In recombinant methods, internal or terminal fragments of apolypeptide can be generated by removing one or more nucleotides fromone end (for a terminal fragment) or both ends (for an internalfragment) of a DNA sequence which encodes for the isolated TWEAKpolypeptide. Expression of the mutagenized DNA produces polypeptidefragments. Digestion with “end nibbling” endonucleases can also generateDNAs which encode an array of fragments. DNAs which encode fragments ofa protein can also be generated by random shearing, restrictiondigestion, or a combination or both. Protein fragments can be generateddirectly from intact proteins. Peptides can be cleaved specifically byproteolytic enzymes, including, but not limited to plasmin, thrombin,trypsin, chymotrypsin, or pepsin. Each of these enzymes is specific forthe type of peptide bond it attacks. Trypsin catalyzes the hydrolysis ofpeptide bonds in which the carbonyl group is from a basic amino acid,usually arginine or lysine. Pepsin and chymotrypsin catalyse thehydrolysis of peptide bonds from aromatic amino acids, such astryptophan, tyrosine, and phenylalanine. Alternative sets of cleavedprotein fragments are generated by preventing cleavage at a site whichis suceptible to a proteolytic enzyme. For instance, reaction of theε-amino acid group of lysine with ethyltrifluorothioacetate in mildlybasic solution yields blocked amino acid residues whose adjacent peptidebond is no longer susceptible to hydrolysis by trypsin. Proteins can bemodified to create peptide linkages that are susceptible to proteolyticenzymes. For instance, alkylation of cysteine residues withβ-haloethylamines yields peptide linkages that are hydrolyzed by trypsin(Lindley, (1956) Nature 178, 647). In addition, chemical reagents thatcleave peptide chains at specific residues can be used. For example,cyanogen bromide cleaves peptides at methionine residues (Gross andWitkip, (1961) J. Am. Chem. Soc. 83, 1510). Thus, by treating proteinswith various combinations of modifiers, proteolytic enzymes and/orchemical reagents, the proteins may be divided into fragments of adesired length with no overlap of the fragments, or divided intooverlapping fragments of a desired length.

Fragments can also be synthesized chemically using techniques known inthe art such as the Merrifield solid phase F moc or t-Boc chemistry.Merrifield, Recent Progress in Hormone Research 23: 451 (1967).

B. Production of Altered DNA and Peptide Sequences: Random Methods

Amino acid sequence variants of a protein can be prepared by randommutagenesis of DNA which encodes the protein or a particular portionthereof. Useful methods include PCR mutagenesis and saturationmutagenesis. A library of random amino acid sequence variants can alsobe generated by the synthesis of a set of degenerate oligonucleotidesequences. Methods of generating amino acid sequence variants of a givenprotein using altered DNA and peptides are well-known in the art. Thefollowing examples of such methods are not intended to limit the scopeof the present invention, but merely serve to illustrate representativetechniques. Persons having ordinary skill in the art will recognize thatother methods are also useful in this regard.

PCR Mutagenesis: See, for example Leung et al., (1989) Technique 1,11-15.

Saturation Mutagenesis: One method is described generally in Mayers etal., (1989) Science 229, 242.

Degenerate Oligonucleotide Mutagenesis: See for example Harang, S. A.,(1983) Tetrahedron 39, 3; Itakura et al., (1984) Ann. Rev. Biochem. 53,323 and Itakura et al., Recombinant DNA, Proc. 3rd Cleveland Symposiumon Macromolecules, pp. 273-289 (A. G. Walton, ed.), Elsevier, Amsterdam,1981.

C. Production of Altered DNA and Peptide Sequences: Directed Methods

Non-random, or directed, mutagenesis provides specific sequences ormutations in specific portions of a polynucleotide sequence that encodesan isolated polypeptide, to provide variants which include deletions,insertions, or substitutions of residues of the known amino acidsequence of the isolated polypeptide. The mutation sites may be modifiedindividually or in series, for instance by: (1) substituting first withconserved amino acids and then with more radical choices depending onthe results achieved; (2) deleting the target residue; or (3) insertingresidues of the same or a different class adjacent to the located site,or combinations of options 1-3.

Clearly, such site-directed methods are one way in which an N-terminalcysteine (or a functional equivalent) can be introduced into a givenpolypeptide sequence to provide the attachment site for a hydrophobicmoiety.

Alanine scanning Mutagenesis: See Cunningham and Wells, (1989) Science244, 1081-1085).

Oligonucleotide-Mediated Mutagenesis: See, for example, Adelman et al.,(1983) DNA 2, 183.

Cassette Mutagenesis: See Wells et al., (1985) Gene 34, 315.

Combinatorial Mutagenesis: See, for example, Ladner et al., W088/06630

Methods of Treatment

The method of the present invention are useful as a treatment indiseases where enhanced angiogenic activity is desirable to promoteneovascularization. Such diseases and conditions include: myocardialischemic conditions (e.g., myocardial infarction, improve blood flow inpatients with coronary artery disease suffering from myocardial ischemiaor inadequate blood flow to areas other than the heart such as inperipheral vascular disease, where decreased blood flow is a problem,revascularization of necrotic tissue, for example of the myocardiumafter an infarction or an angioplasty, angina, heart transplants,vascular grafts, and reopening vessels to improve vascularization,perfusion, collagenization and organization of said lesions), woundhealing, and tissue and organ transplantations (e.g., enhancement ofautologous or heterologous microvascular transplantation). Promotion ofwound healing includes healing of incisions, bone repair, burn healing,post-infarction repair in myocardial injury, healing of gastric ulcersand other ulcers of the gastrointestinal tract and generally inpromoting the formation, maintenance and repair of tissue.Neovascularization of grafted or transplanted tissue is alsocontemplated, especially in subjects suffering from vascularinsufficiency, such as diabetic patients.

As a general matter, the methods of the present invention may beutilized for any mammalian subject needing modulation of angiogenesis.Mammalian subjects which may be treated according to the methods of theinvention include, but are not limited to, human subjects or patients.In addition, however, the invention may be employed in the treatment ofdomesticated mammals which are maintained as human companions (e.g.,dogs, cats, horses), which have significant commercial value (e.g.,dairy cows, beef cattle, sporting animals), which have significantscientific value (e.g., captive or free specimens of endangeredspecies), or which otherwise have value. In addition, as a generalmatter, the subjects for treatment with the methods of the presentinvention need not present indications for treatment with the agents ofthe invention other than those indications associated with need formodulation of angiogenesis. That is, the subjects for treatment areexpected to be otherwise free of indications for treatment with theTWEAK therapeutic agents of the invention.

One of ordinary skill in the medical or veterinary arts is trained torecognize subjects which may need modulation of angiogenesis. Inparticular, clinical and non-clinical trials, as well as accumulatedexperience, relating to the presently disclosed and other methods oftreatment, are expected to inform the skilled practitioner in decidingwhether a given subject is in need of modulation and whether anyparticular treatment is best suited to the subject's needs, includingtreatment according to the present invention.

Accordingly, the methods of this invention may employ TWEAK agonists orbiologically active portions thereof, and angiogenic factors, to promoteangiogenesis, such as, to repair damage of myocardial tissue as a resultof myocardial infarction. Such methods may also include the repair ofthe cardiac vascular system after ischemia including the growth ofcollateral vasculature. Methods utilizing TWEAK agonists and angiogenicfactors may be employed to stimulate the growth of transplanted tissueand collateral vasculature where coronary bypass surgery is performed.Methods may also treat damaged vascular tissue as a result of coronaryartery disease and peripheral or central nervous system vascular diseaseor ischemia.

Methods of the invention may also promote wound healing, particularly tore-vascularize damaged tissues or stimulate collateral blood flow duringischemia and where new capillary angiogenesis is desired. Other methodsof the invention may be employed to treat full-thickness wounds such asdermal ulcers, including pressure sores, venous ulcers, and diabeticulcers. In addition, methods employing TWEAK therapeutics may beemployed to treat full-thickness burns and injuries where a skin graftor flap is used to repair such burns and injuries. Such TWEAK agonistsand angiogenic factors may also be employed for use in plastic surgery,for example, for the repair of lacerations, burns, or other trauma. Inurology, methods of the invention may assist in recovery of erectilefunction.

Since angiogenesis is important in keeping wounds clean andnon-infected, methods may be employed in association with surgery andfollowing the repair of cuts. They may also be employed for thetreatment of abdominal wounds where there is a high risk of infection.Methods using TWEAK therapeutics described herein may be employed forthe promotion of endothelialization in vascular graft surgery. In thecase of vascular grafts using either transplanted or synthetic material,TWEAK agonists and angiogenic factors can be applied to the surface ofthe graft or at the junction to promote the growth of vascular smoothmuscle and adventitial cells in conjunction with endothelial cells.

Methods of the invention may also be employed to coat artificialprostheses or natural organs which are to be transplanted in the body tominimize rejection of the transplanted material and to stimulatevascularization of the transplanted materials and may also be employedfor vascular tissue repair, for example, that occurring duringarteriosclerosis and required following balloon angioplasty wherevascular tissues are damaged. Specifically, methods of the invention maybe employed to promote recovery from arterial wall injury and therebyinhibit restenosis.

Nucleic acid sequences encoding TWEAK agonists may also be employed forin vitro purposes related to scientific research, synthesis of DNA andmanufacture of DNA vectors, and for the production of diagnostics andtherapeutics to treat human disease. For example, methods of theinvention may involve in vitro culturing of vascular smooth musclecells, fibroblasts, hematopoietic cells, muscle, myotendonous junction,bone or cartilage- derived cells and other mesenchymal cells, where aTWEAK therapeutic is added to the conditional medium in a concentrationfrom 10 ng/ml to 20 ug/ml.

These therapeutic agents may be administered by any route which iscompatible with the particular agent employed. The therapeutic agents ofthe invention may be provided to an individual by any suitable means,preferably directly (e.g., locally, as by injection or topicaladministration to a tissue locus) or systemically (e.g., parenterally ororally). Where the agent is to be provided parenterally, such as byintravenous, intraarterial, subcutaneous, or intramuscular,administration, the agent preferably comprises part of an aqueoussolution. The solution is physiologically acceptable so that in additionto delivery of the desired agent to the subject, the solution does nototherwise adversely affect the subject's electrolyte and/or volumebalance. The aqueous medium for the therapeutic may comprise normalphysiologic saline (e.g., 9.85% NaCl, 0.15M, pH 7-7.4).

The therapeutics are preferably administered as a sterile pharmaceuticalcomposition containing a pharmaceutically acceptable carrier, which maybe any of the numerous well known carriers, such as water, saline,phosphate buffered saline, dextrose, glycerol, ethanol, and the like, orcombinations thereof. The compounds of the present invention may be usedin the form of pharmaceutically acceptable salts derived from inorganicor organic acids and bases. Included among such acid salts are thefollowing: acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.Base salts include ammonium salts, alkali metal salts, such as sodiumand potassium salts, alkaline earth metal salts, such as calcium andmagnesium salts, salts with organic bases, such as dicyclohexylaminesalts, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine and saltswith amino acids such as arginine, lysine, and so forth. Also, the basicnitrogen-containing groups can be quatemized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chloride,bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl,dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl,myristyl and stearyl chlorides, bromides and iodides, aralkyl halides,such as benzyl and phenethyl bromides and others. Water or oil-solubleor dispersible products are thereby obtained.

Pharmaceutical compositions of TWEAK agonists and angiogenic factorscomprise any of the compounds of the present invention, orpharmaceutically acceptable derivatives thereof, together with anypharmaceutically acceptable carrier. The term “carrier” as used hereinincludes acceptable adjuvants and vehicles. Pharmaceutically acceptablecarriers that may be used in the pharmaceutical compositions of thisinvention include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Injection Delivery

According to this invention, the pharmaceutical compositions may be inthe form of a sterile injectable preparation, for example a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as do naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant.

Controlled release administration of a particular therapeutic may beuseful. For example, the therapeutic may be administered usingintravenous infusion, an implantable osmotic pump, a transdermal patch,liposomes, or other modes of administration. In one embodiment, a pumpmay be used [Langer et al., eds., Medical Applications of ControlledRelease, CRC Pres., Boca Raton, Fla. (1974); Sefton, CRC Crit. Ref.Biomed. Eng., 14:201 (1987); Buchwald et al., Surgery, 88:507 (1980);Saudek et al., N. Engl. J. Med., 321:574 (1989)]. In another embodiment,polymeric materials can be used [see, Langer, 1974, supra; Sefton, 1987,supra; Smolen et al., eds., Controlled Drug Bioavailability, DrugProduct Design and Performance, Wiley, N.Y. (1984); Ranger et al., J.Macromol. Sci. Rev. Macromol. Chem., 23:61 (1983); see also Levy et al.,Science, 228:190 (1985); During et al., Ann. Neurol., 25:351 (1989);Howard et al., J. Neurosurg., 71:105 (1989)]. In yet another embodiment,a controlled release system can be placed in proximity of thetherapeutic target, e.g., a tumor, thus requiring only a fraction of thesystemic dose [see. e.g., Goodson, in Medical Applications of ControlledRelease, vol. 2, pp. 115-138 (1984)]. Other controlled release systemsare discussed in the review by Langer, Science, 249:1527-1533 (1990). Inanother embodiment, the therapeutic compound can be delivered in avesicle, in particular a liposome (see Langer, 1990, supra); Treat etal., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, pp. 317-327; see generally id.).

Oral Delivery

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Martin, Chapter 89, 1990, supra, which is hereinincorporated by reference. Solid dosage forms include tablets, capsules,pills, troches or lozenges, cachets or pellets. Also, liposomal orproteinoid encapsulation may be used to formulate the presentcompositions (as, for example, proteinoid microspheres reported in U.S.Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, in Modern Pharmaceutics, Chapter 10,Banker and Rhodes ed., (1979), herein incorporated by reference. Ingeneral, the formulation will include the therapeutic (or chemicallymodified form), and inert ingredients which allow for protection againstthe stomach environment, and release of the biologically active materialin the intestine.

For the protein (or derivative) the location of release may be thestomach, the small intestine (the duodenum, the jejunem, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the protein (or derivative) or byrelease of the biologically active material beyond the stomachenvironment, such as in the intestine. To ensure full gastricresistance, a coating impermeable to at least pH 5.0 is essential.Examples of the more common inert ingredients that are used as entericcoatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films. A coating or mixture of coatingscan also be used on tablets, which are not intended for protectionagainst the stomach. This can include sugar coatings, or coatings whichmake the tablet easier to swallow. Capsules may consist of a hard shell(such as gelatin) for delivery of dry therapeutic i.e. powder; forliquid forms, a soft gelatin shell may be used. The shell material ofcachets could be thick starch or other edible paper. For pills,lozenges, molded tablets or tablet triturates, moist massing techniquescan be used.

The therapeutic can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression. Colorants andflavoring agents may all be included. For example, the protein (orderivative) may be formulated (such as by liposome or microsphereencapsulation) and then further contained within an edible product, suchas a refrigerated beverage containing colorants and flavoring agents.One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, alpha-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell. Disintegrants may be included inthe formulation of the therapeutic into a solid dosage form. Materialsused as disintegrants include but are not limited to starch includingthe commercial disintegrant based on starch, Explotab. Sodium starchglycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin,sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose,natural sponge and bentonite may all be used. Another form of thedisintegrants are the insoluble cationic exchange resins. Powdered gumsmay be used as disintegrants and as binders and these can includepowdered gums such as agar, Karaya or tragacanth. Alginic acid and itssodium salt are also useful as disintegrants. Binders may be used tohold the therapeutic agent together to form a hard tablet and includematerials from natural products such as acacia, tragacanth, starch andgelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) andcarboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) andhydroxypropylmethyl cellulose (HPMC) could both be used in alcoholicsolutions to granulate the therapeutic. An antifrictional agent may beincluded in the formulation of the therapeutic to prevent stickingduring the formulation process. Lubricants may be used as a layerbetween the therapeutic and the die wall, and these can include but arenot limited to: stearic acid including its magnesium and calcium salts,polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils andwaxes. Soluble lubricants may also be used such as sodium laurylsulfate, magnesium lauryl sulfate, polyethylene glycol of variousmolecular weights, and Carbowax 4000 and 6000. Glidants that mightimprove the flow properties of the drug during formulation and to aidrearrangement during compression might be added. The glidants mayinclude starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment, asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios. Additiveswhich potentially enhance uptake of the protein (or derivative) are forinstance the fatty acids oleic acid, linoleic acid and linolenic acid.

Pulmonary Delivery

Also contemplated herein is pulmonary delivery of the present proteins(or derivatives thereof). The protein (or derivative) is delivered tothe lungs of a mammal while inhaling and traverses across the lungepithelial lining to the blood-stream. Other reports of this includeAdjei et al., Pharmaceutical Research, 7(6):565-569 (1990); Adjei etal., International Journal of Pharmaceutics, 63:135-144 (1990)(leuprolide acetate); Braquet et al., Journal of CardiovascularPharmacology, 13(suppl. 5):143-146 (1989) (endothelin-1); Hubbard etal., Annals of Internal Medicine, 3(3):206-212 (1989) (alpha1-antitrypsin); Smith et al., J. Clin. Invest., 84:1145-1146 (1989)(alpha 1-proteinase); Oswein et al., “Aerosolization of Proteins”,Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,Colo., (March 1990) (recombinant human growth hormone); Debs et al., J.Immunol., 140:3482-3488 (1988) (interferon-gamma and tumor necrosisfactor alpha) and Platz et al., U.S. Pat. No. 5,284,656 (granulocytecolony stimulating factor). Contemplated for use in the practice of thisinvention are a wide range of mechanical devices designed for pulmonarydelivery of therapeutic products, including but not limited tonebulizers, metered-dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered-dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass. All such devices require the use offormulations suitable for the dispensing of protein (or derivative).Typically, each formulation is specific to the type of device employedand may involve the use of an appropriate propellant material, inaddition to the usual diluents, adjuvants and/or carriers useful intherapy. Also, the use of liposomes, microcapsules or microspheres,inclusion complexes, or other types of carriers is contemplated.Chemically modified protein may also be prepared in differentformulations depending on the type of chemical modification or the typeof device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise protein (or derivative) dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per ml of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the protein (or derivative)suspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing protein (or derivative) and mayalso include a bulking agent, such as lactose, sorbitol, sucrose, ormannitol in amounts which facilitate dispersal of the powder from thedevice, e.g., 50 to 90% by weight of the formulation. The protein (orderivative) should most advantageously be prepared in particulate formwith an average particle size of less than 10 mu m (or microns), mostpreferably 0.5 to 5 mu m, for most effective delivery to the distallung.

Dosages

For all of the above molecules, as further studies are conducted,information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, will be able to ascertain the proper dosage.Generally, for injection or infusion, dosage will be between 0.01 mu gof biologically active protein/kg body weight, (calculating the mass ofthe protein alone, without chemical modification), and 10 mg/kg (basedon the same). The dosing schedule may vary, depending on the circulationhalf-life of the protein or derivative used, whether the polypeptide isdelivered by bolus dose or continuous infusion, and the formulationused.

Administration with Other Compounds

For therapy associated with modulating angiogenesis, one may administerthe present TWEAK agonists (or derivatives) and angiogenic factors inconjunction with one or more pharmaceutical compositions used fortreating other clinical complications of the need for angiogenicmodulation, such as those used for treatment of cancer (e.g.,chemotherapeutics), cachexia, high blood pressure, high cholesterol, andother adverse conditions. Administration may be simultaneous or may bein seriatim.

Nucleic Acid-Based Therapeutic Treatment

Nucleic acid sequences encoding a TWEAK agonist could be introduced intohuman tumor or blood vessel cells to develop gene therapy.

In one embodiment, a nucleic acid sequence encoding a TWEAK agonist isintroduced in vivo in a viral vector. Such vectors include an attenuatedor defective DNA virus, such as but not limited to herpes simplex virus(HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus,adeno-associated virus (AAV), and the like. Defective viruses, whichentirely or almost entirely lack viral genes, are preferred. Defectivevirus is not infective after introduction into a cell. Use of defectiveviral vectors allows for administration to cells in a specific,localized area, without concern that the vector can infect other cells.Thus, adipose tissue can be specifically targeted. Examples ofparticular vectors include, but are not limited to, a defective herpesvirus 1 (HSV1) vector [Kaplitt et al., Molec. Cell. Neurosci., 2:320-330(1991)], an attenuated adenovirus vector, such as the vector describedby Stratford-Perricaudet et al., J. Clin. Invest., 90:626-630 (1992),and a defective adeno-associted virus vector [Samulski et al., J.Virol., 61:3096-3101 (1987); Samulski et al., J. Virol., 63:3822-3828(1989)]. In another embodiment, the nucleic acid can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al., Cell, 33:153 (1983); Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol., 62:1120 (1988); Temin et al., U.S. Pat. No. 5,124,263;International Patent Publication No. WO 95/07358, published Mar. 16,1995, by Dougherty et al.; and Kuo et al., Blood, 82:845 (1993).Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker [Felgneret al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987); see Mackey etal., Proc. Natl. Acad. Sci. USA, 85:8027-8031 (1988)]. The use ofcationic lipids may promote encapsulation of negatively charged nucleicacids, and also promote fusion with negatively charged cell membranes[Felgner et al., Science, 337:387-388 (1989)]. The use of lipofection tointroduce exogenous genes into specific organs in vivo has certainpractical advantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly advantageous in a tissuewith cellular heterogeneity, such as the pancreas, liver, kidney, andbrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting (see Mackey et al., 1988, supra). Targetedpeptides, e.g., hormones or neurotransmitters, and proteins such asantibodies, or non-peptide molecules could be coupled to liposomeschemically.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fuision, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (see, e.g., Wu et al., J. Biol. Chem.,267:963-967 (1992); Wu et al., J. Biol. Chem., 263:14621-14624 (1988);Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar.15, 1990).

It is also possible to introduce the vector in vivo in conjunction witha catheter or other device. See Vale et al., 1999; Komowski et al.,2000.

EXAMPLES

The following examples illustrate aspects of the present invention butshould not be construed as limitations. The symbols and convention usedin these examples are consistent with those used in contemporary medicaland scientific literature

Experimental Procedures:

Cells—Human Umbilical Vein Endothelial Cells (HUVEC) were obtained fromCell System Corporation (CS-C) (Kirkland, Wash.) or Clonetics (SanDiego, Calif.) and Human Pulmonary Artery Endothelial Cells (HPAEC),Human Lung Microvascular Endothelial cells (HMVEC-L) and Human DermalMicrovascular Endothelial cells (HMVEC-D) were purchased from Clonetics.HUVEC were routinely passaged in CS-C Medium and used in experimentsuntil passage seven. The other primary cells were routinely passaged inMicrovascular Endothelial Cell Growth Medium-2 (EGM2-MV) (Clonetics). ECBasal Medium (EBM) containing 2% fetal bovine serum (FBS), defined as“basal media”, and EBM containing 2% FBS and supplier growthsupplements, defined as “complete media”, were used in proliferation,migration and immunofluorescent staining experiments. EC Basal Medium 2EBM-2) and supplier growth supplements were used in the capillary tubeformation assay as specified.

Reagents and Antibodies—Recombinant human bFGF was obtained as a growthsupplier supplement (Clonetics), bFGF also was purchased from R&DSystems (Minneapolis, Minn.) and Sigma (St.Louis, Mo.). Annexin V-FITCwas from Pharmingen (San Diego, Calif.), propidium iodide (PI) fromSigma (St.Louis, Mo.), rabbit anti-human Flk-1 and rabbit anti-Flt-1antibodies from Research Diagnostics Inc. (Flanders, N.J.), mouseanti-Flg monoclonal antibody (mAb) from Chemicon (Temecula, Calif.),mouse anti-human-β₃, mouse anti-human-β₁ (clone LIA ½) and ratanti-human α_(v) mAbs from Immunotech (Westbrook, Me.), mouse anti-humanα₅ from Pharmingen (San Diego, Calif.), mouse anti-human α₁ (cloneAJH10) from Biogen (Gotwals P. J, et al., 1999. Biochem. 38:8280-8288),Phycoerythrin (PE)-conjugated donkey anti-rabbit IgG, goat anti-mouseIgG and donkey anti-rat IgG from Jackson Immunoresearch Labs Inc. (WestGroove, Pa.), biotin-conjugated anti-FLAG from Eastman Kodak Company(New Haven, Conn.), and RPE-Streptavidin from Southern BiotechnologyAssociates, Inc. (Birmingham, Ala.). Soluble CD40L was prepared atBiogen as previously described (Karpusas, M., et al., 1995. Structure3:1426-xxx).

TWEAK-specific mAbs BE.B3 and AB.D3 were generated in Armenian hamstersby immunizing with soluble human TWEAK protein and standard hybridomageneration procedures. The ability of AB.D3 to bind to human and murineTWEAK and BE.B3 to bind to human TWEAK was demonstrated in an ELISAassay using recombinant soluble TWEAK proteins immobilized on 96 wellmicrotiter plates. The blocking activity of AB.D3 was demonstrated bythe ability of this mAb but not BE.B3 to inhibit soluble FLAG-taggedhuman TWEAK binding to HT29 cells in a FACS analysis. BE.B3 wasbiotinylated prepared using ImmunoPure Biotinylation kits following themanufacturer's protocol (Pierce, Rockford, Ill.). A hamster control Ig(clone Ha4/8-3.1) was obtained from the American Type Culture Collectionand mAb purified from culture supernatant by Protein A Fast Flow column(Pharmacia, Piscataway, N.J.).

Recombinant Soluble Human TWEAK protein—Soluble expression construct formyc-tagged human TWEAK was constructed as previously described(Chicheportiche, Y., et al. 1997. J. Biol. Chem. 272:32401-32410).Flag-tagged and nontagged forms also were made. These soluble forms ofTWEAK were expressed in yeast, Pichia pastoris strain GS115, usingstandard conditions.

Proliferation Assays—HUVEC were plated in 96-well microtiter plates atsubconfluence (4000 cells per well) and cultured overnight in CS-CMedium without addition of supplier growth supplements. Media wasreplaced with complete Media, or with basal media as defined above.Cells were cultured in basal media with or without TWEAK (100 ng/ml),bFGF using a 1/500- 1/1000 dilution of bFGF growth supplement(Clonetics) or 1 ng/ml (R&D Systems), VEGF (10 ng/ml) or combinations ofthese factors. Where indicated, 10 μg/ml anti-TWEAK mAbs AB.D3, BE.B3 orhamster control Ig Ha4/8 also were added. Cells were incubated at 37° C.with 5% CO₂ for three days and proliferation was measured by pulsingwith ³H-Thymidine for the last 10 hours of culture. Cell-boundradioactivity was measured with a Betaplate™ (EG&G Wallac, Gaithersburg,Md.).

Analysis of Apoptosis—HUVEC seeded in 6-well plates at a density of1.2×10⁵ cells per well were incubated over night in CS-C Medium withoutsupplier growth supplements. Media was replaced with complete media, orwith basal media with or without TWEAK (200 ng/ml), bFGF (1 ng/ml) orcombinations of these factors and cells were cultured for 24 hours.Cells were washed with Phosphate buffered saline (PBS) and detached byincubation with dispase (CS-C) for 15 minutes at 37° C. followed byreplacement with PBS containing 5 mM EDTA and 0.1% BSA for 15 minutes at37° C. After an additional wash in PBS, cells were stained withFITC-Annexin-V and 5 μg/ml Propidium Iodide according to the supplier(Pharmingen). Fluorescence was analyzed within the hour usingFACStar^(PLUS) (Becton Dickinson, San Jose, Calif.).

Endothelial Wound Repair Assay—A standard wound repair assay wasemployed as previously described (Bussolino F., et al, 1991. J. Clin.Invest. 87:986-991). In brief, a confluent monolayer of HUVEC was grownin CS-C Medium in 35×10 mm cell culture dishes with 2 mm grids (NalgeNunc International, Naperville, Ill.). The monolayer was wounded by twoperpendicular strokes across the diameter of the dish with a 1 mm tip(Morales D. E., et al., 1995. Circulation 91:755-763). Dislodged cellswere aspirated and plates were rinsed with PBS. Cells were cultured incomplete media, or in fresh basal media with or without TWEAK (200ng/ml), bFGF ( 1/1000 or 1 ng/ml), VEGF (10 ng/ml) or combinations ofthese and were incubated for 18 hours at 37° C. with 5% CO₂ at whichtime plates were fixed with 1% paraformaldehyde and stained with HarrisHematoxylin (Sigma, St. Louis, Mo.). Wound repair was quantified byvisually counting the number of grids in which the gap was obscured bymigrating cells. This number was divided by the total number of gridsthat aligned the wound and results were expressed as mean percentagewound repair +/−SEM.

Immunofluorescent staining—HUVEC were cultured in basal media with orwithout TWEAK (200 ng/ml), bFGF (1 ng/ml) or both factors for 24 hours.Cells were detached as described above and stained with 10 μg/ml primaryantibody in 200 μl PBS containing 0.1% bovine serum albumin and 0.02%NaN₃ for 20 minutes at 4° C. Following washes with the same buffer, thePE-conjugated detection antibodies were added at concentrations asspecified by the manufacturer for an additional 15 minutes at 4° C.Cells were analyzed for TWEAK binding by incubation with TWEAK taggedeither with flag or with myc. Binding was detected with eitherbiotinylated mouse anti-flag antibody or biotinylated BE.B3 andstreptavidin-PE. Cold competition was performed with non-tagged TWEAKand blocking was performed with the AB.D3 mAb.

Capillary tube formation assay—Capillary tube formation by ECs wasanalyzed using a three-dimensional fibrin matrix gel assay based on amethod previously described (Mach, F., et al., 1999. Am. J. Pathol.154:229-239). Briefly, 4 mg/ml plasminogen free human fibrinogen(Calbiochem, San Diego, Calif.) was dissolved in serum free EBM-2 mediawith heparin and polymixin B both at 1 μg/ml (Sigma) as well as all ofthe supplier supplements except for VEGF and bFGF. The fibrin solutionwas filtered-sterilized and fibrin matrices were prepared by addingthrombin (20-50 milliunits/ml) (Sigma) and distributing 300 ul per wellin 24-well plates. ECs at appropriate concentrations (4×10⁴ cells/cm²for HUVEC and HPAEC and 8×10⁴/cm² for HMVEC-L and HMVEC-D) were thenseeded onto the gel surfaces and overlayered with EBM-2 media as aboveand 5% FBS in the presence or absence of TWEAK, bFGF, sCD40L orcombinations of these factors as specified. After 72 hours of culture,phase-contrast photomicrographs of the gel surface were taken. Gels weretransferred from original wells to new wells and fixed with 10% ethanolfor 10 minutes and then with 4% paraformaldehyde. Gels were crosssectioned for analysis and photographs taken.

Example 1 TWEAK Enhances bFGF-Dependent Proliferation

The effect of TWEAK on EC functions was investigated by examining ECproliferation in cultures treated with TWEAK alone and in combinationwith another angiogenic growth factors. Human Umbilical Vein EC (HUVEC)were cultured in basal media in the presence or absence of bFGF (FIG.1). Addition of TWEAK induced no significant proliferation of ECs. Bycontrast, cells cultured with TWEAK and an optimal concentration of bFGFdisplayed a significantly enhanced proliferative response compared tocells cultured in the presence of bFGF alone. The degree ofproliferation achieved was comparable to or greater than that of ECscultured in complete media. Similar results were obtained using bFGF at1 ng/ml. The synergistic activity of TWEAK with bFGF was completelyinhibited by anti-TWEAK mAb AB.D3 suggesting that the effect of TWEAKwas specific, whereas there was no inhibition by a anti-TWEAK mAb BE.B3or an irrelevant control Ig. In addition, no enhancement was seen withrecombinant soluble APRIL, another TNF ligand (data not shown). Theexperimental conditions for the results shown in FIG. 1 are describedhere in detail. HUVEC were cultured in complete media or in basal media.TWEAK (100 ng/ml), bFGF ( 1/500 dilution) or combinations of thesefactors were added to basal media as indicated for 3 days andproliferation measured by ³H-thymidine incorporation. In FIG. 1, datashown are the mean value +/−SD of triplicate wells. These results arerepresentative of 4 independent experiments wherein proliferation inbFGF+TWEAK-treated cultures was significantly different from that ofcultures with bFGF alone, TWEAK alone and basal media (P values<0.05),and the difference between cultures in basal media with and withoutTWEAK was not significant. In addition to growth factors, blockinganti-TWEAKmAb AB.D3, nonblocking anti-TWEAK mAb BE.B3, and an irrelevanthamster control Ig Ha4/8 (10 ug/ml) were added where indicated. Resultsare representative of one of two independent experiments.

Example 2 Enhancement of EC Proliferation by TWEAK with bFGF Is Not Dueto Decreased Cell Death

The apparent enhancement of HUVEC proliferation by the TWEAK/bFGFcombination could be due to increased cell division or decreased celldeath. In order to address the mechanism, HUVEC cultured in basal mediawith or without TWEAK, bFGF or both were analyzed to determine thefrequency of apoptotic cells. Annexin V staining was employed to detectcells undergoing apoptosis and propidium iodide (PI) dye exclusion todetect viable cells. Cultures treated with the combination of TWEAK andbFGF exhibited percentages of viable, apoptotic and dead cells that werecomparable to those of cultures treated with bFGF alone. Thesepercentages are shown in FIG. 2, in quadrants 3, 4 and 2 respectively.Similar results were obtained in two additional experiments whereincells with subdiploid DNA content were quantified (11% in bFGF and 11%in bFGF/TWEAK treated cultures). Thus, the enhancement by TWEAK ofbFGF-dependent proliferation is not due to decreased cell death.Nevertheless it is noteworthy that TWEAK alone decreased the frequencyof apoptotic cells from 22% to 14%. This pattern also was observed intwo independent experiments, wherein the percentage of cells withsubdiploid DNA were on average 18+/−1% and 9+/−0% in the absence andpresence of TWEAK, respectively.

The experimental conditions for the results in FIG. 2 are described indetail here. HUVEC cultured for 24 hours in basal media with or withoutTWEAK (200 ng/ml), bFGF (1 ng/ml) or both cytokines were stained withFITC-Annexin-V (x-axis) for apoptotic cells and by PI dye exclusion forviability (y-axis). FIG. 2 shows the percentage of viable, apoptotic anddead cells in quadrants 3, 4, and 2, respectively.

Example 3 TWEAK Enhances bFGF-Dependent HUVEC Migration

The ability of TWEAK to effect EC migration was evaluated in thepresence and absence of other angiogenic factors. Confluent HUVECmonolayers were wounded and EC migration was monitored within the first18 hours by determining the degree of wound repair. Addition of TWEAK orbFGF to basal media induced a low level of wound repair, however, thiswas not significantly greater than that observed with basal media alone.By contrast, cultures treated with both TWEAK and bFGF were repaired toa significantly greater degree than cultures in basal media and witheither agent alone, and were similar to those in complete media. HUVECswere recovered from the cultures and counted in order to determinewhether or not any increase in cell number had occurred over the courseof the experiment. In all treatment groups, cell recoveries werecomparable (data not shown) supporting that the combinatorial effect ofTWEAK and bFGF was at the level of cell migration.

The experimental conditions for the results shown in FIG. 3 aredescribed here in detail. Confluent HUVEC monolayers treated with TWEAK(200 ng/ml), bFGF (1 ng/ml or 1/1000 dilution), and combinations ofthese factors, were wounded and repair measured after 18 hours ofculture. FIG. 3 shows the average of 4 experiments +/−SEM, with repairinduced by bFGF+TWEAK significantly different from that induced byeither alone or basal media (P values<0.05).

Example 4 Effect of TWEAK Is Not Mediated by Modulation of Growth FactorReceptors or Integrins

Integrins, especially α_(v)β₃, α₁β₁ and α₂β₁ facilitate cell migrationthrough extracellular matrix and also regulate cell survival andintracellular signaling required for the response to angiogenic factors(Eliceiri, B. and Cheresh, D. A., 1999. J. Clin. Invest. 103:1227-1230;Senger, D. R., et al., 1997. Proc. Natl. Acad. Sci. 94:13612-13617).Therefore, we aimed to determine whether or not TWEAK modulated growthfactor receptors or integrins expressed on ECs. VEGF receptors Flk-1 andFlt-1 and bFGF receptor Flg were expressed at very low levels on HUVECscultured in basal media. As a positive control, these receptor-specificmAbs showed strong staining on human dermal microvascular EC (HMVEC-D).Consistent with the study by Lynch et al (11), we found no change in theexpression of VEGF receptors Flk-1 and Flt-1 in TWEAK treated cultures,nor were there changes in VEGF receptor expression in cultures treatedwith bFGF or the TWEAK/bFGF combination. In addition, we found thatTWEAK treatment did not alter the level of the bFGF receptor Flg or ofintegrins α_(v), α₁, α₅, β₁, and β₃.

Example 5 TWEAK Induces EC Morphogenesis

A key event in the angiogenic process is the organization of invadingECs into capillary tubes. The effect of TWEAK on this morphogenic stepwas measured with EC seeded onto the surface of three-dimensional fibringels in the presence or absence of bFGF. We found no effect of TWEAK onthe EC monolayer, while an optimal concentration of bFGF promoted cellinvasion and organization of EC into cords. The addition of TWEAK tobFBF induced clear morphological changes in the EC monolayer. Similarresults were obtained with several different EC types, including HUVECs,human pulmonary artery ECs (HPAEC), human lung microvascular EC(HMVEC-L) and HMVEC-D. In addition, cross-sectional analysis of thesegels revealed that the addition of TWEAK to bFBF induced the structuralorganization of invading ECs into tubes with lumens. CD40L, another TNFmember, had no effect either alone or in combination with bFGF. ThusTWEAK synergizes with bFGF to induce the morphogenesis of capillarylumens. The results are shown in FIG. 4.

1-6. (canceled)
 7. A method for enhancing endothelial cell proliferation in an in vitro culture comprising adding to said culture, a formulation consisting essentially of a synergistically effective amount of a TWEAK agonist and an angiogenic factor.
 8. A method for enhancing angiogenic activity in a mammal to promote neovascularization comprising the step of administering to said mammal a formulation consisting essentially of a synergistically effective amount of a TWEAK agonist and an angiogenic factor sufficient to promote neovascularization.
 9. The methods according to claims 7 or 8, wherein the angiogenic factor is selected from the group consisting of hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), angiopoietin 1, angiopoietin 2, and monocyte chemotactic protein-1 (MCP-1).
 10. The methods according to claims 7 or 8, wherein the angiogenic factor is bFGF.
 11. The method of claim 8, wherein said method is used in the treatment of a myocardial ischemic condition.
 12. The method of claim 8, wherein said method is used to promote wound healing.
 13. The method of claim 8, wherein said method is used in the treatment of dermal ulcers, lacerations, burns, or other dermal trauma in said mammal.
 14. The method of claim 8, wherein said method is used to promote growth of collateral vasculature after ischemia or recovery of erectile function in said mammal.
 15. A method for in vitro culturing of mammalian cells in an in vitro culture comprising adding to said culture a formulation comprising a TWEAK agonist and an angiogenic factor.
 16. The method of claim 15, wherein the mammalian cells are selected from the group consisting of vascular smooth muscle cells, fibroblasts, hematopoietic cells, muscle, myotendonous junction, bone-derived cells, cartilage-derived cells, and other mesenchymal cells.
 17. The method of claim 15, wherein the TWEAK agonist and the angiogenic factor are present in a synergistically effective amount.
 18. The method of claim 15, wherein the formulation consists of a TWEAK agonist and an angiogenic factor.
 19. The method of claim 15, wherein the TWEAK agonist is TWEAK.
 20. The method of claim 15, wherein the angiogenic factor is selected from the group consisting of hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), angiopoietin 1, angiopoietin 2, and monocyte chemotactic protein-1 (MCP-1).
 21. The method of claim 15, wherein the angiogenic factor is bFGF.
 22. The method of claim 15, wherein the TWEAK agonist is TWEAK and the angiogenic factor is bFGF.
 23. A formulation comprising a TWEAK agonist and an angiogenic factor.
 24. The formulation of claim 23, wherein the formulation consists of a TWEAK agonist and an angiogenic factor.
 25. The formulation of claim 23, wherein the TWEAK agonist and the angiogenic factor are present in a synergistically effective amount.
 26. The formulation of claim 23, wherein the TWEAK agonist is TWEAK.
 27. The formulation of claim 23, wherein the angiogenic factor is selected from the group consisting of hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), angiopoietin 1, angiopoietin 2, and monocyte chemotactic protein-1 (MCP-1).
 28. The formulation of claim 23, wherein the angiogenic factor is bFGF.
 29. The formulation of claim 23, wherein the TWEAK agonist is TWEAK and the angiogenic factor is bFGF.
 30. A cell culture comprising: mammalian cells, a cell culture medium, and a combination of a TWEAK agonist and an angiogenic factor, wherein the combination is an amount sufficient to promote angiogenesis.
 31. The cell culture of claim 30, wherein the mammalian cells are endothelial cells, vascular smooth muscle cells, fibroblasts, hematopoietic cells, muscle, myotendonous junction, bone-derived cells, cartilage-derived cells, and other mesenchymal cells
 32. The cell culture of claim 30, wherein the TWEAK agonist and the angiogenic factor are present in a synergistically effective amount.
 33. The cell culture of claim 30, wherein the TWEAK agonist is TWEAK.
 34. The cell culture of claim 30, wherein the angiogenic factor is bFGF.
 35. The cell culture of claim 30, wherein the TWEAK agonist is TWEAK and the angiogenic factor is bFGF.
 36. A method of identifying inhibitors of TWEAK and bFGF mediated cellular proliferation, the method comprising: culturing endothelial cells in the presence of TWEAK and bFGF; culturing endothelial cells in the presence of TWEAK, bFGF, and a test compound; and comparing the amount of endothelial cell proliferation in the cell culture containing the test compound to the amount of proliferation in the cell culture not containing with the test compound, wherein a decrease in proliferation in the culture treated with the test compound indicates that the test compound is an inhibitor of proliferation.
 37. The method of claim 36, wherein the test compound is an antibody. 